Cellular liquid biopsy provides unique chances for disease monitoring, preclinical model generation and therapy adjustment in rare salivary gland cancer patients.

While cell-free liquid biopsy (cfLB) approaches provide simple and inexpensive disease monitoring, cell-based liquid biopsy (cLB) may enable additional molecular genetic assessment of systemic disease heterogeneity and preclinical model development. We investigated 71 blood samples of 62 patients with various advanced cancer types and subjected enriched circulating tumor cells (CTCs) to organoid culture conditions. CTC-derived tumoroid models were characterized by DNA/RNA sequencing and immunohistochemistry, as well as functional drug testing. Results were linked to molecular features of primary tumors, metastases, and CTCs; CTC enumeration was linked to disease progression. Of 52 samples with positive CTC counts (≥1) from eight different cancer types, only CTCs from two salivary gland cancer (SGC) patients formed tumoroid cultures (P = 0.0005). Longitudinal CTC enumeration of one SGC patient closely reflected disease progression during treatment and revealed metastatic relapse earlier than clinical imaging. Multiomics analysis and functional in vitro drug testing identified potential resistance mechanisms and drug vulnerabilities. We conclude that cLB might add a functional dimension (to the genetic approaches) in the personalized management of rare, difficult-to-treat cancers such as SGC.

Hepatic Bone Morphogenetic Protein and Activin Membrane-Bound Inhibitor Levels Decline in Hepatitis C but Are Not Associated with Progression of Hepatocellular Carcinoma.

BACKGROUND/OBJECTIVES: Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is an antagonist of transforming growth factor (TGF)-ß type 1 signaling. BAMBI functions as an anti-fibrotic protein and exerts pro- as well as anti-cancerogenic activities. Our study aimed to correlate hepatocyte BAMBI protein levels in hepatocellular carcinoma (HCC) with T stage, lymph node invasion, vessel invasion, grading, tumor size and Union for International Cancer Control (UICC) stage, as well as with liver inflammation and fibrosis stages. METHODS: Hepatocyte BAMBI protein expression was assessed by immunohistochemistry in HCC tissues of 320 patients and non-tumor tissues of 51 patients. RESULTS: In the HCC tissues of the whole cohort and sex-specific analysis, BAMBI protein was not related to T stage, vessel invasion, lymph node invasion, histologic grade, UICC stage and tumor size. Accordingly, BAMBI was not associated with overall survival, recurrence-free and metastasis-free survival. BAMBI protein levels in tumor and non-tumor tissues were not related to inflammation and fibrosis grade. BAMBI protein levels in HCC tissues and non-tumor tissues from HCC patients, which were analyzed by immunoblot in a small cohort and by immunohistochemistry in the tissues of patients described above, were similar. Notably, BAMBI protein was low-abundant in HCC tissues of hepatitis C virus (HCV) compared to hepatitis B virus (HBV)-infected patients with comparable disease severity. Immunoblot analysis revealed reduced BAMBI protein in non-tumor tissues of patients with HCV in comparison to patients with HBV and normal human liver tissues. CONCLUSIONS: In summary, this analysis showed that hepatocyte BAMBI protein levels of patients with HCC are related to HCV infection rather than the severity of the underlying liver disease and cancer staging.

Comparative Analysis of Mitochondria Surrounding the Intercalated Discs in Heart Diseases-An Ultrastructural Pilot Study.

BACKGROUND: Mitochondria play a crucial role in adapting to fluctuating energy demands, particularly in various heart diseases. This study investigates mitochondrial morphology near intercalated discs in left ventricular (LV) heart tissues, comparing samples from patients with sinus rhythm (SR), atrial fibrillation (AF), dilated cardiomyopathy (DCM), and ischemic cardiomyopathy (ICM). METHODS: Transmission electron microscopy was used to analyze mitochondria within 0-3.5 µm and 3.5-7 µm of intercalated discs in 9 SR, 10 AF, 9 DCM, and 8 ICM patient samples. Parameters included mean size in µm2 and elongation, count, percental mitochondrial area in the measuring frame, and a conglomeration score. RESULTS: AF patients exhibited higher counts of small mitochondria in the LV myocardium, resembling SR. DCM and ICM groups had fewer, larger, and often hydropic mitochondria. Accumulation rates and percental mitochondrial area were similar across groups. Significant positive correlations existed between other defects/size and hydropic mitochondria and between count/area and conglomeration score, while negative correlations between count and size/other defects and between hydropic mitochondria and count could be seen as well. CONCLUSION: Mitochondrial parameters in the LV myocardium of AF patients were similar to those of SR patients, while DCM and ICM displayed distinct changes, including a decrease in number, an increase in size, and compromised mitochondrial morphology. Further research is needed to fully elucidate the pathophysiological role of mitochondrial morphology in different heart diseases, providing deeper insights into potential therapeutic targets and interventions.

Elastography-The New Standard in the Assessment of Fibrosis After Pediatric Liver Transplantation?

BACKGROUND: The development of graft fibrosis after pediatric liver transplantation (PLT) remains a major concern as it can lead to graft failure and ultimately graft loss. Elastography is a non-invasive method to assess liver fibrosis, but its role in the posttransplant setting is unclear. The aim of our study was to evaluate shear wave elastography (SWE) in the assessment of liver fibrosis after PLT, including split-liver recipients. METHODS: We retrospectively analyzed data from PLT recipients who underwent surveillance liver biopsy and concurrent 2D-SWE during the study period from April 2018 to July 2021. Spearman's correlation was used to compare histologic fibrosis stages with liver stiffness measurements (LSM) by 2D-SWE. AUROC analysis was performed to evaluate the performance. One sample t-test was used to compare results with reference values of healthy children. RESULTS: 62 cases were included. 29% showed histologic fibrosis. LSM by 2D-SWE were feasible in all children regardless of age or graft type. There was a significant correlation between LSM and fibrosis stage for all three scoring systems used (Ishak, p = 0.003; METAVIR, p = 0.005; LAF Score, p = 0.003). Patients with a history of biliary complications had increased liver stiffness (p = 0.015). The AUROC of 2D-SWE for predicting significant liver graft fibrosis was 0.81. Liver stiffness after PLT without graft fibrosis was higher than in healthy subjects, but comparable to that in children with chronic liver disease without fibrosis. CONCLUSION: 2D-SWE can reliably detect children with significant liver graft fibrosis, even in split-liver recipients. This study demonstrates the value of a non-invasive tool for fibrosis staging after PLT. 2D-SWE has the potential to improve long-term outcomes after PLT and to reduce the number of surveillance liver biopsies. But elastography is not a substitute for liver biopsy.

Expression of the plasma membrane citrate carrier (pmCiC) in human cancerous tissues-correlation with tumour aggressiveness.

We have recently shown that cancer cells of various origins take up extracellular citrate through the plasma membrane citrate carrier (pmCiC), a specific plasma membrane citrate transporter. Extracellular citrate is required to support cancer cell metabolism, in particular fatty acid synthesis, mitochondrial activity, protein synthesis and histone acetylation. In addition, cancer cells tend to acquire a metastatic phenotype in the presence of extracellular citrate. Our recent study also showed that cancer-associated stromal cells synthesise and release citrate and that this process is controlled by cancer cells. In the present study, we evaluated the expression of pmCiC, fibroblast activation protein-α (FAP) and the angiogenesis marker cluster of differentiation 31 (CD31) in human cancer tissues of different origins. In the cohort studied, we found no correlation between disease stage and the expression of FAP or CD31. However, we have identified a clear correlation between pmCiC expression in cancer cells and cancer-associated stroma with tumour stage. It can be concluded that pmCiC is increased in cancer cells and in cancer-supporting cells in the tumour microenvironment at the later stages of cancer development, particularly at the metastatic sites. Therefore, pmCiC expression has the potential to serve as a prognostic marker, although further studies are needed.

Soluble CD46 as a diagnostic marker of hepatic steatosis.

BACKGROUND: The increasing prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) incurs substantial morbidity, mortality and healthcare costs. Detection and clinical intervention at early stages of disease improves prognosis; however, we are currently limited by a lack of reliable diagnostic tests for population screening and monitoring responses to therapy. To address this unmet need, we investigated human invariant Natural Killer T cell (iNKT) activation by fat-loaded hepatocytes, leading to the discovery that circulating soluble CD46 (sCD46) levels accurately predict hepatic steatosis. METHODS: sCD46 in plasma was measured using a newly developed immuno-competition assay in two independent cohorts: Prospective living liver donors (n = 156; male = 66, female = 90) and patients with liver tumours (n = 91; male = 58, female = 33). sCD46 levels were statistically evaluated as a predictor of hepatic steatosis. FINDINGS: Interleukin-4-secreting (IL-4+) iNKT cells were over-represented amongst intrahepatic lymphocytes isolated from resected human liver samples. IL-4+ iNKT cells preferentially developed in cocultures with a fat-loaded, hepatocyte-like cell line, HepaRG. This was attributed to induction of matrix metalloproteases (MMP) in fat-loaded HepaRG cells and primary human liver organoids, which led to indiscriminate cleavage of immune receptors. Loss of cell-surface CD46 resulted in unrepressed differentiation of IL-4+ iNKT cells. sCD46 levels were elevated in patients with hepatic steatosis. Discriminatory cut-off values for plasma sCD46 were found that accurately classified patients according to histological steatosis grade. INTERPRETATION: sCD46 is a reliable clinical marker of hepatic steatosis, which can be conveniently and non-invasively measured in serum and plasma samples, raising the possibility of using sCD46 levels as a diagnostic method for detecting or grading hepatic steatosis. FUNDING: F.B. was supported by the Else Kröner Foundation (Award 2016_kolleg.14). G.G. was supported by the Bristol Myers Squibb Foundation for Immuno-Oncology (Award FA-19-009). N.S. was supported by a Wellcome Trust Fellowship (211113/A/18/Z). J.A.H. received funding from the European Union's Horizon 2020 research and innovation programme (Award 860003). J.M.W. received funding from the Else Kröner Foundation (Award 2015_A10).

Histological assessment of cardiac amyloidosis in patients undergoing transcatheter aortic valve replacement.

AIMS: Studies have reported a strongly varying co-prevalence of aortic stenosis (AS) and cardiac amyloidosis (CA). We sought to histologically determine the co-prevalence of AS and CA in patients undergoing transcatheter aortic valve replacement (TAVR). Consequently, we aimed to derive an algorithm to identify cases in which to suspect the co-prevalence of AS and CA. METHODS AND RESULTS: In this prospective, monocentric study, endomyocardial biopsies of 162 patients undergoing TAVR between January 2017 and March 2021 at the University Medical Centre Göttingen were analysed by one pathologist blinded to clinical data using haematoxylin-eosin staining, Elastica van Gieson staining, and Congo red staining of endomyocardial biopsies. CA was identified in only eight patients (4.9%). CA patients had significantly higher N-terminal pro-brain natriuretic peptide (NT-proBNP) levels (4356.20 vs. 1938.00 ng/L, P = 0.034), a lower voltage-to-mass ratio (0.73 vs. 1.46 × 10-2 mVm2/g, P = 0.022), and lower transaortic gradients (Pmean 17.5 vs. 38.0 mmHg, P = 0.004) than AS patients. Concomitant CA was associated with a higher prevalence of post-procedural acute kidney injury (50.0% vs. 13.1%, P = 0.018) and sudden cardiac death [SCD; P (log-rank test) = 0.017]. Following propensity score matching, 184 proteins were analysed to identify serum biomarkers of concomitant CA. CA patients expressed lower levels of chymotrypsin (P = 0.018) and carboxypeptidase 1 (P = 0.027). We propose an algorithm using commonly documented parameters-stroke volume index, ejection fraction, NT-proBNP levels, posterior wall thickness, and QRS voltage-to-mass ratio-to screen for CA in AS patients, reaching a sensitivity of 66.6% with a specificity of 98.1%. CONCLUSIONS: The co-prevalence of AS and CA was lower than expected, at 4.9%. Despite excellent 1 year mortality, AS + CA patients died significantly more often from SCD. We propose a multimodal algorithm to facilitate more effective screening for CA containing parameters commonly documented during clinical routine. Proteomic biomarkers may yield additional information in the future.

Surgical Treatment and Outcome of Ovarian Cancer Patients With Liver Metastases: Experience of a Tertiary Hepatic and Peritoneal Surface Malignancy Center.

BACKGROUND/AIM: The aim of this study was to describe and evaluate the patterns, perioperative outcomes, and survival rates of patients subjected to hepatic resections for ovarian-derived liver metastasis as part of cytoreductive surgery with or without hyperthermic intraperitoneal chemotherapy (HIPEC). Furthermore, we investigated two subgroups of tumor patterns: hematogenous liver metastasis and infiltrative liver metastatic spread. PATIENTS AND METHODS: A retrospective study was conducted. Patients from a University Tertiary Hepatic and Peritoneal Surface Malignancy Center with primary or recurrent ovarian cancer, who underwent liver resection as part of cytoreductive surgery between January 1992 and December 2022, were included. RESULTS: Data from 35 patients were analyzed. Both median overall survival (OS) and disease-specific survival (DSS) were 24.97 months. In a multivariate setting, the combined effect of age, peritoneal carcinomatosis index, body mass index, hematogenous liver metastasis vs. infiltrative spread types, and HIPEC (HR=0.2372; 95%CI=0.0719-0.7823; p=0.0181) over OS was tested. Survival analysis revealed no differences between the two metastatic spread types (OS: p=0.9720; DSS: p=0.9610). Younger age (p=0.0301), splenectomy (p=0.0320), lesser omentectomy (p=0.0178), and right upper quadrant peritonectomy (p=0.0373) were more characteristic for those patients with infiltrative liver metastatic spread. CONCLUSION: Complete cytoreductive surgery, including hepatic resection is a feasible approach with or without additional HIPEC, which may provide survival benefit for patients with advanced and/or recurrent ovarian cancer. If metastatic and infiltrative liver involvement is suspected, liver-specific imaging is recommended.

Targeting the microenvironment in the treatment of arteriovenous malformations.

Extracranial arteriovenous malformations (AVMs) are regarded as rare diseases and are prone to complications such as pain, bleeding, relentless growth, and high volume of shunted blood. Due to the high vascular pressure endothelial cells of AVMs are exposed to mechanical stress. To control symptoms and lesion growth pharmacological treatment strategies are urgently needed in addition to surgery and interventional radiology. AVM cells were isolated from three patients and exposed to cyclic mechanical stretching for 24 h. Thalidomide and bevacizumab, both VEGF inhibitors, were tested for their ability to prevent the formation of circular networks and proliferation of CD31+ endothelial AVM cells. Furthermore, the effect of thalidomide and bevacizumab on stretched endothelial AVM cells was evaluated. In response to mechanical stress, VEGF gene and protein expression increased in patient AVM endothelial cells. Thalidomide and bevacizumab reduced endothelial AVM cell proliferation. Bevacizumab inhibited circular network formation of endothelial AVM cells and lowered VEGF gene and protein expression, even though the cells were exposed to mechanical stress. With promising in vitro results, bevacizumab was used to treat three patients with unresectable AVMs or to prevent regrowth after incomplete resection. Bevacizumab controlled bleeding, pulsation, and pain over the follow up of eight months with no patient-reported side effects. Overall, mechanical stress increases VEGF expression in the microenvironment of AVM cells. The monoclonal VEGF antibody bevacizumab alleviates this effect, prevents circular network formation and proliferation of AVM endothelial cells in vitro. The clinical application of bevacizumab in AVM treatment demonstrates effective symptom control with no side effects.

Hepatitis E virus infection of transplanted kidneys.

Immunocompromised patients are at risk of chronic hepatitis E (HEV) infection. Recurrent T cell and borderline rejections in a pediatric patient with high HEV copy numbers led us to study HEV infection within renal transplants. To investigate the frequency of renal HEV infection in transplanted patients, 15 samples from patients with contemporaneous diagnoses of HEV infection were identified at our center. Ten samples had sufficient residual paraffin tissue for immunofluorescence (IF) and RNA-fluorescence-in-situ-hybridization (RNA-FISH). The biopsy of the pediatric index patient was additionally sufficient for tissue polymerase chain reaction and electron microscopy. HEV RNA was detected in paraffin tissue of the index patient by tissue polymerase chain reaction. Subsequently, HEV infection was localized in tubular epithelial cells by IF, RNA-FISH, and electron microscopy. One additional biopsy from an adult was positive for HEV by RNA-FISH and IF. Focal IF positivity for HEV peptide was observed in 7 additional allografts. Ribavirin therapy was not successful in the pediatric index patient; after relapse, ribavirin is still administered. In the second patient, successful elimination of HEV was achieved after short-course ribavirin therapy. HEV infection is an important differential diagnosis for T cell rejection within transplanted kidneys. Immunostaining of HEV peptide does not necessarily prove acute infection. RNA-FISH seems to be a reliable method to localize HEV.

Deep learning-based phenotyping reclassifies combined hepatocellular-cholangiocarcinoma.

Primary liver cancer arises either from hepatocytic or biliary lineage cells, giving rise to hepatocellular carcinoma (HCC) or intrahepatic cholangiocarcinoma (ICCA). Combined hepatocellular- cholangiocarcinomas (cHCC-CCA) exhibit equivocal or mixed features of both, causing diagnostic uncertainty and difficulty in determining proper management. Here, we perform a comprehensive deep learning-based phenotyping of multiple cohorts of patients. We show that deep learning can reproduce the diagnosis of HCC vs. CCA with a high performance. We analyze a series of 405 cHCC-CCA patients and demonstrate that the model can reclassify the tumors as HCC or ICCA, and that the predictions are consistent with clinical outcomes, genetic alterations and in situ spatial gene expression profiling. This type of approach could improve treatment decisions and ultimately clinical outcome for patients with rare and biphenotypic cancers such as cHCC-CCA.

Identification of DUSP4/6 overexpression as a potential rheostat to NRAS-induced hepatocarcinogenesis.

BACKGROUND: Upregulation of the mitogen-activated protein kinase (MAPK) cascade is common in hepatocellular carcinoma (HCC). Neuroblastoma RAS viral oncogene homolog (NRAS) is mutated in a small percentage of HCC and is hitherto considered insufficient for hepatocarcinogenesis. We aimed to characterize the process of N-Ras-dependent carcinogenesis in the liver and to identify potential therapeutic vulnerabilities. METHODS: NRAS V12 plasmid was delivered into the mouse liver via hydrodynamic tail vein injection (HTVI). The resulting tumours, preneoplastic lesions, and normal tissue were characterized by NanoString® gene expression analysis, Western Blot, and Immunohistochemistry (IHC). The results were further confirmed by in vitro analyses of HCC cell lines. RESULTS: HTVI with NRAS V12 plasmid resulted in the gradual formation of preneoplastic and neoplastic lesions in the liver three months post-injection. These lesions mostly showed characteristics of HCC, with some exceptions of spindle cell/ cholangiocellular differentiation. Progressive upregulation of the RAS/RAF/MEK/ERK signalling was detectable in the lesions by Western Blot and IHC. NanoString® gene expression analysis of preneoplastic and tumorous tissue revealed a gradual overexpression of the cancer stem cell marker CD133 and Dual Specificity Phosphatases 4 and 6 (DUSP4/6). In vitro, transfection of HCC cell lines with NRAS V12 plasmid resulted in a coherent upregulation of DUSP4 and DUSP6. Paradoxically, this upregulation in PLC/PRF/5 cells was accompanied by a downregulation of phosphorylated extracellular-signal-regulated kinase (pERK), suggesting an overshooting compensation. Silencing of DUSP4 and DUSP6 increased proliferation in HCC cell lines. CONCLUSIONS: Contrary to prior assumptions, the G12V NRAS mutant form is sufficient to elicit hepatocarcinogenesis in the mouse. Furthermore, the upregulation of the MAPK cascade was paralleled by the overexpression of DUSP4, DUSP6, and CD133 in vivo and in vitro. Therefore, DUSP4 and DUSP6 might fine-tune the excessive MAPK activation, a mechanism that can potentially be harnessed therapeutically.

Expression of G protein-coupled receptor GPR19 in normal and neoplastic human tissues.

Little is known about the expression of the orphan G protein-coupled receptor GPR19 at the protein level. Therefore, we developed a rabbit antibody, targeting human GPR19. After verification of the antibody specificity using GPR19-expressing cell lines and a GPR19-specific siRNA, the antibody was used for immunohistochemical staining of a variety of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. In normal tissues, GPR19 expression was detected in a distinct cell population within the cortex, in single cells of the pancreatic islets, in intestinal ganglia, gastric chief cells, and in endocrine cells of the bronchial tract, the gastrointestinal tract, and the prostate. Among the 30 different tumour entities investigated, strong GPR19 expression was found in adenocarcinomas, typical and atypical carcinoids of the lung, and small cell lung cancer. To a lesser extent, the receptor was also present in large cell neuroendocrine carcinomas of the lung, medullary thyroid carcinomas, parathyroid adenomas, pheochromocytomas, and a subpopulation of pancreatic neuroendocrine neoplasms. In lung tumours, a negative correlation with the expression of the proliferation marker Ki-67 and a positive interrelationship with patient survival was observed. Overall, our results indicate that in adenocarcinomas and neuroendocrine tumours of the lung GPR19 may serve as a suitable diagnostic or therapeutic target.

Chemerin and Chemokine-like Receptor 1 Expression Are Associated with Hepatocellular Carcinoma Progression in European Patients.

The chemoattractant protein chemerin is protective in experimental hepatocellular carcinoma (HCC), and high expression in HCC tissues of Asian patients was related to a favorable prognosis. Studies from Asia found reduced expression of chemerin in HCC compared to para-tumor tissues while our previous analysis observed the opposite. Aim of this study was to correlate chemerin expression in HCC tissues with disease severity of European patients Hepatocyte chemerin protein expression was assessed by immunohistochemistry in HCC tissue of 383 patients, and was low in 24%, moderate in 49% and high in 27%. High chemerin protein in the HCC tissues was related to the T stage, vessel invasion, histologic grade, Union for International Cancer Control (UICC) stage and tumor size. Chemokine-like receptor 1 (CMKLR1) is a functional chemerin receptor. CMKLR1 protein in hepatocytes was low expressed in HCC tissues of 36%, moderate in tissues of 32% and high in 32% of the HCCs. Tumor CMKLR1 was associated with the T stage, vessel invasion, histologic grade and UICC stage. Notably, sex-specific analysis revealed that associations of chemerin and CMKLR1 expression with HCC progression were significant in males but not in females. The tumor chemerin and CMKLR1 protein expression were not related to steatosis, inflammation and fibrosis grades. In summary, chemerin as well as CMKLR1 protein were related to disease severity of European HCC patients, and this was significant in males. This observation is in contrast to Asian patients where higher chemerin in the tumors was protective. Current analysis provides evidence for ethnicity and sex-related differences of tumor expressed chemerin and HCC severity.

Anti-DNA-IgM Favors the Detection of NET-Associated Extracellular DNA.

During inflammatory responses, neutrophils enter the sites of attack where they execute various defense mechanisms. They (I) phagocytose microorganisms, (II) degranulate to release cytokines, (III) recruit various immune cells by cell-type specific chemokines, (IV) secrete anti-microbials including lactoferrin, lysozyme, defensins and reactive oxygen species, and (V) release DNA as neutrophil extracellular traps (NETs). The latter originates from mitochondria as well as from decondensed nuclei. This is easily detected in cultured cells by staining of DNA with specific dyes. However, in tissues sections the very high fluorescence signals emitted from the condensed nuclear DNA hamper the detection of the widespread, extranuclear DNA of the NETs. In contrast, when we employ anti-DNA-IgM antibodies, they are unable to penetrate deep into the tightly packed DNA of the nucleus, and we observe a robust signal for the extended DNA patches of the NETs. To validate anti-DNA-IgM, we additionally stained the sections for the NET-markers histone H2B, myeloperoxidase, citrullinated histone H3, and neutrophil elastase. Altogether, we have described a fast one-step procedure for the detection of NETs in tissue sections, which provides new perspectives to characterize neutrophil-associated immune reactions in disease.

Phenotypic Characterization of Colorectal Liver Metastases: Capsule versus No Capsule and the Potential Role of Epithelial Mesenchymal Transition.

Background: Colorectal liver metastases (CRLM) can be encased in a fibrous capsule separating cancer from normal liver tissue, which correlates with increased patient survival. This study investigated the cellular and molecular components of capsule formation and the possible role of epithelial mesenchymal transition (EMT). Methods: From 222 patients with CRLM, 84 patients (37.8%) were categorized to have CRLM encased with a capsule. A total of 34 CRLM from 34 selected patients was analyzed in detail by EMT pathway-profiling and custom PCR arrays to identify differences in gene expression between CRLM with (n = 20) and without capsule (n = 14). In parallel, those 34 CRLM were used to analyze 16 gene products at the metastasis margin via immunohistochemistry. Results: Encapsulated CRLM showed an elevated expression of signal transduction pathways and effector molecules involved in EMT. E-cadherin and keratin-19 were more prevalent, and transcription as well as translation (immunohistochemistry) of pGSK-3-ß, SOX10, tomoregulin-1, and caldesmon were increased. By contrast, the loss of E-cadherin and the prevalence of snail-1 were increased in CRLM without capsule. Collagen I and III and versican were identified as capsule components with extracellular matrix fibers running concentrically around the malignant tissue and parallel to the invasive front. Caldesmon was also demonstrated as a capsule constituent. Conclusions: The fibrous capsule around CRLM can be produced by cells with mesenchymal characteristics. It functions as a protective border by both the features of fiber architecture and the inhibition of invasive growth through EMT recruiting mesenchymal cells such as myofibroblasts by transformation of surrounding epithelial or even carcinoma cells. By contrast, EMT demonstrated in non-encapsulated CRLM may lead to a more mesenchymal, mobile, and tissue-destructive carcinoma cell phenotype and facilitate malignant spread.

Aberrant fucosylation sustains the NOTCH and EGFR/NF-κB pathways and has a prognostic value in human intrahepatic cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a lethal malignancy, with increasing incidence worldwide and limited therapeutic options. Aberrant protein glycosylation is a hallmark of cancer. Here, we thoroughly investigated the possible involvement of fucosylation in cholangiocarcinogenesis. APPROACH AND RESULTS: We discovered that the levels of global fucosylation and members of the fucosylation pathway are ubiquitously upregulated in human iCCA tissues compared to nontumorous surrounding livers and normal biliary cells. In addition, total fucosylation levels correlate with poor patients' prognosis. Furthermore, fucosylation inhibition following 6-alkynylfucose (6AF) administration triggered a dose-dependent decrease in the proliferation and migration of iCCA cell lines. Notably, adding fucose to the cell medium annulled these effects. At the molecular level, 6AF administration or small interfering RNA-mediated silencing of GDP-L-fucose synthetase (FX) and the GDP-fucose transmembrane transporter (SLC35C1), both pivotal players of cellular fucosylation, decreased NOTCH activity, NOTCH1/Jagged1 interaction, NOTCH receptors, and related target genes in iCCA cell lines. In the same cells, EGFR, nuclear factor kappa-light-chain-enhancer of activated B cells p65, and Bcl-xL protein levels diminished, whereas IκBα (a critical cellular NF-κB inhibitor) increased after FX/SLC35C1 knockdown or 6AF administration. In the chick chorioallantoic membrane assay, 6AF treatment profoundly suppresses the growth of iCCA cells. CONCLUSIONS: Elevated global fucosylation characterizes human iCCA, contributing to cell growth and migration through the upregulation of the NOTCH and EGFR/NF-κB pathways. Thus, aberrant fucosylation is a novel pathogenetic player and a potential therapeutic target for human iCCA.

Early Subcellular Hepatocellular Alterations in Mice Post Hydrodynamic Transfection: An Explorative Study.

Hydrodynamic transfection (HT) or hydrodynamic tail vein injection (HTVi) is among the leading technique that is used to deliver plasmid genes mainly into the liver of live mice or rats. The DNA constructs are composed of coupled plasmids, while one contains the gene of interest that stably integrate into the hepatocyte genome with help of the other consisting sleeping beauty transposase system. The rapid injection of a large volume of DNA-solution through the tail vein induces an acute cardiac congestion that refluxed into the liver, mainly in acinus zone 3, also found through our EM study. Although, HT mediated hydrodynamic force can permeabilizes the fenestrated sinusoidal endothelium of liver, but the mechanism of plasmid incorporation into the hepatocytes remains unclear. Therefore, in the present study, we have hydrodynamically injected 2 mL volume of empty plasmid (transposon vector) or saline solution (control) into the tail vein of anesthetized C57BL/6J/129Sv mice. Liver tissue was resected at different time points from two animal group conditions, i.e., one time point per animal (1, 5, 10-20, 60 min or 24 and 48 hrs after HT) or multiple time points per animal (0, 1, 2, 5, 10, 20 min) and quickly fixed with buffered 4% osmium tetroxide. The tissues fed with only saline solution was also resected and fixed in the similar way. EM evaluation from the liver ultrathin sections reveals that swiftly after 1 min, the hepatocytes near to the central venule in the acinus zone 3 shows cytoplasmic membrane-bound vesicles. Such vesicles increased in both numbers and size to vacuoles and precisely often found in the proximity to the nucleus. Further, EM affirm these vacuoles are also optically empty and do not contain any electron dense material. Although, some of the other hepatocytes reveals sign of cell damage including swollen mitochondria, dilated endoplasmic reticulum, Golgi apparatus and disrupted plasma membrane, but most of the hepatocytes appeared normal. The ultrastructural findings in the mice injected with empty vector or saline injected control mice were similar. Therefore, we have interpreted the vacuole formation as nonspecific endocytosis without specific interactions at the plasma membrane.

Long-Term Simulation of Microgravity Induces Changes in Gene Expression in Breast Cancer Cells.

Microgravity changes the gene expression pattern in various cell types. This study focuses on the breast cancer cell lines MCF-7 (less invasive) and MDA-MB-231 (triple-negative, highly invasive). The cells were cultured for 14 days under simulated microgravity (s-µg) conditions using a random positioning machine (RPM). We investigated cytoskeletal and extracellular matrix (ECM) factors as well as focal adhesion (FA) and the transmembrane proteins involved in different cellular signaling pathways (MAPK, PAM and VEGF). The mRNA expressions of 24 genes of interest (TUBB, ACTB, COL1A1, COL4A5, LAMA3, ITGB1, CD44, VEGF, FLK1, EGFR, SRC, FAK1, RAF1, AKT1, ERK1, MAPK14, MAP2K1, MTOR, RICTOR, VCL, PXN, CDKN1, CTNNA1 and CTNNB1) were determined by quantitative real-time PCR (qPCR) and studied using STRING interaction analysis. Histochemical staining was carried out to investigate the morphology of the adherent cells (ADs) and the multicellular spheroids (MCSs) after RPM exposure. To better understand this experimental model in the context of breast cancer patients, a weighted gene co-expression network analysis (WGCNA) was conducted to obtain the expression profiles of 35 breast cell lines from the HMS LINCS Database. The qPCR-verified genes were searched in the mammalian phenotype database and the human genome-wide association studies (GWAS) Catalog. The results demonstrated the positive association between the real metastatic microtumor environment and MCSs with respect to the extracellular matrix, cytoskeleton, morphology, different cellular signaling pathway key proteins and several other components. In summary, the microgravity-engineered three-dimensional MCS model can be utilized to study breast cancer cell behavior and to assess the therapeutic efficacies of drugs against breast cancer in the future.